PROJECT SUMMARY Drug abuse and drug addiction are very common in HIV/AIDS patients. Abused drugs are well known cofactors which exacerbate HIV/AIDS pathology, including HIV associated neurological disorders (HAND). In detail, drugs act on multiple cell types in brain, including astrocytes. Astrocytes are most abundant brain cells and they play very important roles in maintaining brain homeostasis. Astrocytes can be infected by HIV and latently infected astrocytes are one of HIV brain reservoirs. Morphine, cocaine and methamphetamine are most abused drugs and they influence brain cells at many points, including modification of cell chromatin structure. In addition, abused drugs often work together with HIV Tat protein on epigenetic regulation and chromatin modification of HIV provirus in HIV/AIDS patients. Recently clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique emerges as one efficient gene editing tool in mammalian cells. However, studies also found Cas9 editing is affected by chromatin structure. Currently only limited studies have applied CRISPR/Cas9 technique to eliminate HIV provirus in HIV infected cells or animal models. Astrocyte specific gene editing has not been investigated due to lack of cell models. Furthermore, there are no reports on drug impacts on Cas9 gene editing of HIV provirus. Our lab recently have established a latent HIV astrocyte model which could indicate CRIPSR/Cas9 gene editing efficiency by fluorescence microscopy, plate reading and cell genomic DNA PCR. In preliminary data we also established similar type of latent HIV model using primary astrocytes. We will take advantage of astrocyte models to study cocaine impact on CRISPR/Cas9 gene editing of HIV provirus with and without HIV Tat protein. Furthermore, in preliminary data, we have found that cocaine treatment increase DNA methyltransferase expression using gene array assay; cocaine and HIV Tat protein could affect Cas9 gene editing of HIV provirus in latent astrocytes. Based on these data, this proposal hypothesize that abused drug cocaine impacts CRISPR/Cas9 gene editing of HIV provirus in latent astrocytes. This impact works through the modification of chromatin structure in infected cells. HIV Tat protein is a cofactor in such impact. In this proposal, we set up two aims to explore the interaction between abused drug cocaine and Cas9 gene editing. Aim 1. To determine the impact of cocaine and HIV Tat protein on CRISPR/Cas9 gene editing of HIV provirus in latent astrocytes and to validate the findings in latent primary astrocytes. Aim 2. To investigate the molecular mechanism of cocaine impact on CRISPR/Cas9 gene editing with focus on DNA methylation, histone acetylation and methylation. The long term goal is to develop therapeutic CRISPR/Cas9 formulations targeting both HIV provirus and cocaine abuse.